The Mitochondrial Ca
2
+
Uniporter MCU Is Essential for
Glucose-Induced ATP Increases in Pancreatic
b
-Cells
Andrei I. Tarasov1, Francesca Semplici1, Magalie A. Ravier1,2, Elisa A. Bellomo1, Timothy J. Pullen1, Patrick Gilon3, Israel Sekler4, Rosario Rizzuto5, Guy A. Rutter1*
1Section of Cell Biology, Division of Diabetes Endocrinology and Metabolism, Department of Medicine, Imperial College London, London, United Kingdom,2Institut de Ge´nomique Fonctionnelle, INSERM U661, CNRS UMR5203, Universite´ Montpellier I et II, Montpellier, France,3Pole of Endocrinology, Diabetes and Nutrition, Faculty of Medicine, Universite´ Catholique de Louvain, Brussels, Belgium, 4Department of Physiology, Faculty of Health Sciences, Ben Gurion University, Beer-Sheva, Israel,
5Department of Biomedical Sciences, University of Padua, Padua, Italy
Abstract
Glucose induces insulin release from pancreaticb-cells by stimulating ATP synthesis, membrane depolarisation and Ca2+ influx. As well as activating ATP-consuming processes, cytosolic Ca2+increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca2+transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca2+ uptake into these organelles, or to examine the impact onb-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca2+and ATP/ADP ratio in single primary mouseb-cells, using recombinant targeted (PericamorPerceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca2+uniporter, MCU, is required for depolarisation-induced mitochondrial Ca2+ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na+-Ca2+exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca2+uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca2+accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreaticb-cells, and becomes defective in conditions mimicking the diabetic milieu.
Citation:Tarasov AI, Semplici F, Ravier MA, Bellomo EA, Pullen TJ, et al. (2012) The Mitochondrial Ca2+
Uniporter MCU Is Essential for Glucose-Induced ATP Increases in Pancreaticb-Cells. PLoS ONE 7(7): e39722. doi:10.1371/journal.pone.0039722
Editor:Valdur Saks, Universite´ Joseph Fourier, France
ReceivedFebruary 27, 2012;AcceptedMay 25, 2012;PublishedJuly 19, 2012
Copyright:ß2012 Tarasov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:Supported by the Wellcome Trust (Programme Grant 081958/Z/07/Z to GAR, Value in People (VIP) award to TJP) and the JDRF (Postdoctoral Fellowship to AIT), Telethon-Italy, Italian Association for Cancer Research, the Italian Ministry of Education (PRIN, FIRB), the Cariparo Foundation and the European Research Council ("mitoCalcium") (for RR). PG is Research Director of the Fonds National de la Recherche Scientifique, Brussels. MAR is Charge´ de Recherches from INSERM, Paris. GAR is a Wellcome Trust Senior Investigator (WT098424AIA) and the holder of a Royal Society Wolfson Research Merit Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: g.rutter@imperial.ac.uk
Introduction
Glucose-induced insulin secretion from pancreatic b-cells is essential to ensure the normal control of blood glucose concen-trations [1]. Defects inb-cell glucose sensitivity [2,3] as well as a decrease inb-cell mass [4] are cardinal aspects of type 2 diabetes mellitus (T2D). A key event in glucose-induced insulin release is the stimulation of mitochondrial oxidative metabolism [5,6]. Enhanced ATP synthesis [7] results in the closure of ATP-sensitive K+
(KATP) channels [8], membrane depolarisation and Ca2+influx via voltage-gated Ca2+
channels, which triggers insulin release [1,9].
In most mammalian cells, mitochondrial oxidative metabolism is thought to be stimulated by Ca2+[10,11] through the activation of intramitochondrial dehydrogenases [12]. This stimulates the supply of reducing equivalents to the respiratory chain [13], and hence ATP synthesis [14]. The above process is thought also to be important in pancreaticb-cells [15] and recent analyses using a mitochondrial Ca2+buffer [14] have suggested that mitochondrial Ca2+accumulation is important for sustained insulin secretion.
The interplay between cytosolic Ca2+
, mitochondrial Ca2+ and ATP synthesis has nonetheless remained enigmatic in theb-cell. In particular, Ca2+
entry into the cytosol, triggered by elevated ATP, is expected to enhance ATP hydrolysis, for example by activating granule exocytosis [16] and Ca2+
ATPases which pump the cation out of the cytosol [17]. The Ca2+
-induced drop in ATP is then predicted to open KATPchannels, thereby arresting Ca2+influx [18]. In addition, Ca2+
has been suggested to induce repolarisation of the plasma membrane by opening Ca2+
-activated K+
channels [19] or depolarising the mitochondrial inner membrane, which decreases the driving force for ATP synthesis by the F1FoATPase [20].
Until very recently, the molecular entities responsible for catalysing mitochondrial Ca2+
uptake have remained unclear in any mammalian cell type. However, two reports in 2011 identified a Ca2+
-selective mitochondrial uniporter, MCU, encoded by the Ccdc109agene [21,22], in a complex with a Ca2+
sensing subunit MICU1 [23], as the likely Ca2+
transporting entity. Conversely, mitochondrial Ca2+
efflux was proposed to be mediated by the Na+
-Ca2+
catalyse mitochondrial Ca2+
transport in theb-cell, and may thus modulate insulin secretion, is currently unknown.
In the present study, we have sought to explore (a) the molecular mechanisms responsible for Ca2+
transfer across the mitochondrial membrane in b-cells and (b) the impact of these changes on cytosolic ATP dynamics and electrical excitability. To these ends, we have deployed a recently-developed, molecularly-addressed GFP-based recombinant probe for mitochondrial Ca2+
([Ca2+ ]mit), 2mt8RP [25], alongside a trappable cytosolic Ca2+
probe (Fura Red) allowing us to image [Ca2+]
cytsimultaneously with [Ca 2+]
mit in individual primary mouse b-cells. These measurements have been combined with perforated patch electrophysiology to allow plasma membrane potential (Vm) to be recorded or controlled without perturbing cellular composition or metabolism [26]. Critically, this approach permits the ready and rapid control of [Ca2+
]cytvia voltage-gated Ca2+channels [27] and thus an analysis of the interplay between [Ca2+
]cytand [Ca2+]mitin real time. In parallel, the novel ATP sensorPerceval[28], based on the bacterial regulatory protein, GlnK1, has been used to monitor the cytosolic ATP/ADP ratio ([ATP/ADP]cyt). These combined approaches have allowed us to characterise the roles of MCU and NCLX as regulators of mitochondrial ATP synthesis in theb-cell.
Results
Glucose induces a monophasic increase in cytosolic Ca2+ but a biphasic increase in cytosolic ATP/ADP ratio
We sought first to determine whether increases in [Ca2+ ]cyt and/or [Ca2+]
mit might influence glucose-induced increases in [ATP/ADP]cyt. The latter parameter was therefore imaged in single mouseb-cells expressing the GFP-based probePerceval[28], which was chiefly localised to the cytosol as expected (Suppl. Fig. S1A). Changes measured with this probe were shown to be unrelated to small alterations in cytosolic pH, and thus largely to reflect [ATP/ADP]cyt (Suppl. Fig. S2A). [Ca2+]cyt was imaged simultaneously in the same cell using the trappable cytosolic/ nuclear probe Fura-Red (Suppl. Fig. S1A) whilst Vm was monitored using patch-clamp in current-clamp mode [3].
b-Cells maintained at low (3 mM) glucose exhibited a resting Vm of 26861 mV (n= 30, from 12 separate islet preparations; pointiin Fig. 1A). An increase in glucose concentration to 17 mM led to a rapid elevation in [ATP/ADP]cyt(Fig. 1A, pointii) and an increase in input resistance, followed by depolarisation of the plasma membrane and a [Ca2+
]cyt rise, as expected. This was closely followed by a drop in [ATP/ADP]cyt(Fig. 1A, iii). The 3364% drop (‘‘trough’’ in Fig. 1B) was, however, transient and [ATP/ADP]cyt quickly recovered and displayed a steady further increase (Fig. 1A, iv). The increase was not associated with any significant decrease in [Ca2+
]cyt, and thus was not likely to reflect a lowering demand for Ca2+
extrusion or other ATP-consuming processes. Furthermore, setting Vm to 270mV via the patch pipette, thus closing voltage-gated Ca2+
channels, led to a prompt decrease in [Ca2+
]cyt (Fig. 1A, v). The application of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP) resulted in an abrupt decrease of [ATP/ ADP]cyt, as expected (Fig. 1A,vi), and an elevation of [Ca2+]cyt, presumably due to a compromise in Ca2+
pumping across the plasma and ER membranes.
Combining data from multiple experiments (n = 30 single cells, Fig. 1B) we were able to observe that high glucose induced an [ATP/ADP]cytelevation inb-cells in two distinct phases (Fig. 1B). A rapid first phase preceded membrane depolarisation and electrical activity, whilst a slower second phase resulted in a larger increase of [ATP/ADP]cyt (Fig. 1B). These changes contrasted
with the essentially monophasic (albeit oscillatory) increases in [Ca2+
]cyt(Fig. 1A).
Cytosolic Ca2+influx is essential for the second phase of cytosolic ATP/ADP ratio increase
To dissect the dependence of the observed ATP increases on cytosolic Ca2+
increases prompted by depolarisation in response to glucose, we measured the changes in [ATP/ADP]cytin response to the sugar while keeping the cell hyperpolarised (Vm= 270mV) using the patch pipette in voltage-clamp mode (as in point v, Fig. 1A). This prevented extracellular Ca2+
from entering the cytosol even at high extracellular glucose.
An increase in glucose from 3 mM to 17 mM resulted in a rapid elevation of [ATP/ADP]cyt, followed by a saturation of the [ATP/ ADP]cytlevel (ii, Fig. 2). Notably, in the absence of Ca2+influx, neither a trough, nor an increase in [ATP/ADP]cyt(see e.g. points iiiandivin Fig. 1A) were observed, suggesting that Ca2+influx is involved in the latter changes. To test this possibility, we imposed forced changes in [Ca2+
]cytwith a train of 10 depolarisations (as given in Suppl. Fig. S2B) and then setting Vmback to270mV (as indicated in the Vmtrace in Fig. 2). The depolarisations triggered rapid and transient [Ca2+
]cytelevation which, in turn, resulted in a transient drop in [ATP/ADP]cyt(iii, Fig. 2) Remarkably, [ATP/ ADP]cytstarted recovering while the depolarisation train was still being applied, at high [Ca2+
]cyt, and this trend continued after Vm had been re-set to270mV and [Ca2+]
cythad decreased (iv, Fig. 2). These experiments indicate that the biphasic behaviour of [ATP/ ADP]cytresponse to glucose is caused by the increase in [Ca2+]cyt which results in a transient drop in [ATP/ADP]cytfollowed by its recovery. The two phases of the glucose-induced increase in [ATP/ADP]cyt can therefore be classified as Ca2+-independent (the one that precedes) and Ca2+
-dependent (the one that follows) Ca2+entry.
We next sought to determine whether the apparent increases in cytosolic ATP/ADP ratio reported with Perceval were associated with the closure of ATP-sensitive K+
channels, as expected. This seemed an important question since fluctuations in ‘‘global’’ cytosolic ATP/ADP differ in some circumstances from those immediately beneath the plasma membrane, as recorded with a targeted luciferase-based probe [7]. The electrophysiological configuration used here allowed us to address this point as follows. While keeping the cell hyperpolarised, at270mV (Fig. 2), we applied small pulses between265 and280 mV, to monitor slow whole-cell current, Im. These pulses were too small to trigger any voltage-gated Ca2+
conductance and therefore had no effect on Ca2+entry. The addition of 17 mM glucose decreased I
mduring the Ca2+
-independent phase of [ATP/ADP]cyt increase (Fig. 2, inset), most likely due to the inhibition of KATPchannels, the main providers of theb-cell conductance (Gm) [29]. Gmthus was found to decrease from the initial value of 0.4360.09 nS/pF to 0.0960.02 nS/pF (n = 12) during the Ca2+
the propagation of the glucose-induced ATP increase from the sub-membrane compartment to the bulk cytosol [7,30].
Glucose induces a sequential increase in [Ca2+]
cytand [Ca2+]
mit
We next explored the possibility that the uptake of Ca2+ by mitochondria may be related to the second phase of [ATP/ ADP]cyt increase, as suggested by earlier experiments in b-cell populations [14]. To explore the temporal relationship between Figure 1. Glucose induces a biphasic increase in cytosolic ATP/ADP ratio.A: The effects of high (17mM) glucose on [ATP/ADP]cyt(reported
withPerceval), [Ca2+]
cyt(Fura-Red) and Vmwere measured in a singleb-cell (representative of n = 30 cells). The voltage down-strokes were elicited by
10 ms 10 pA current injections applied every 20 s to monitor the input resistance which increased upon the elevation of [ATP/ADP]cyt.Inset:
Pseudo-colour images of the patched cell cluster presenting pixel-to-pixel ratios at the time points indicated by arrows (i–vi). ROI is indicated with red oval. Note that a cell expressing high levels ofPerceval(just below the ROI) was deliberately excluded from analysis.B: Characteristic times and amplitudes of glucose-induced [ATP/ADP]cytincrease inb-cells (Fig. 1A; n = 30). The data were normalised to the width of the range of [ATP/ADP]cytchange
(DFmax), measured as the difference inPercevalfluorescence between the peak point at 17 mM glucose and the point corresponding to application of
2mM FCCP. Depolarisation and onset of electrical activity was taken as zero of the time axis. The change in [ATP/ADP]cyt(DF/DFmax) at each point is
significantvsevery other point (p,0.01, Wilcoxon’s paired test). doi:10.1371/journal.pone.0039722.g001
Ca2+
increases in [Ca2+
]cyt and [Ca2+]mit in single b-cells after stimulation with glucose, we used a mitochondrial matrix-targeted fluorescent Ca2+
probe, 2mt8RP [25] (Fig. 3A; Supp. Fig. S1B). At 3 mM glucose, the plasma membrane was hyperpolarised as expected (Vm= 26861 mV, n = 22) and [Ca2+]cytand [Ca2+]mit were stable (Fig. 3B, pointi). Exposure to 17 mM glucose led to an increase in [Ca2+
]cyt(Fig. 3B, ii) which was followed later by an increase in [Ca2+
]mit, presumably reflecting Ca2+ uniporter-mediated uptake (Fig. 3B, iii). [Ca2+
]cyt and [Ca2+]mit reached their maximal amplitudes 4766 s and 134625 s, respectively, after the onset of glucose-induced electrical activity (Fig. 3C).
MCU mediates mitochondrial Ca2+ increases and the second phase of glucose-induced [ATP/ADP]cytincreases
In experiments using an identical configuration to those above, the maximal rate of [ATP/ADP]cyt decrease was observed 106622 s after the first action potential (between pointsiiandiii in Fig. 1A). This observation, and those described for the time course of mitochondrial Ca2+ increases (Fig. 3B, C), are thus consistent with the possibility that mitochondrial Ca2+ accumula-tion (and hence an activaaccumula-tion of oxidative metabolism) plays a role in the regulation of the [ATP/ADP]cyt increase that follows an initial and small Ca2+-induced drop. To test this possibility directly we therefore reduced the expression of the recently-identified mitochondrial Ca2+uniporter, MCU [21,22], inb-cells by
.80% (as assessed by qRT-PCR, not shown) using a lentivirally-delivered shRNA (Fig. 4). Silencing of MCU caused a substantial impairment of apparent Ca2+entry into mitochondria, whilst the imposed cytosolic Ca2+ increases were unaffected (Fig. 4A, B). Importantly, this manipulation also resulted in an alteration of the glucose-induced [ATP/ADP]cytchanges (Fig. 5A, B). Thus, MCU silencing had no effect on the first phase of the glucose-induced
[ATP/ADP]cyt increase, the rise of [Ca2+]cyt or subsequent electrical spiking (Fig. 5A). However, the second (Ca2+
-dependent) phase of the [ATP/ADP]cyt increase, i.e. the [ATP/ADP]cyt recovery, was significantly impaired in theb-cells where MCU expression was reduced (Fig. 5A, B).
To determine whether MCU knock-down might affect mito-chondrial membrane potential (Ym) independently of a Ca2+ increase, we explored the glucose-induced changes in this parameter prior to [Ca2+]
cyt elevation using tetramethyl rhoda-mine, ethyl ester (TMRE). The resting Ym (measured as 212764 mV in control vs 213365 mV in MCU2 cells) and the kinetics of the glucose-induced change (Fig. 5C) were not affected by the knock-down of MCU.
NCLX modulates mitochondrial Ca2+changes Pharmacological inhibition of mitochondrial Na+
-Ca2+ ex-change has been reported to elevate the basal ATP levels in INS-1 cells and primary rat islets [31]. However, the agent used (CGP37157) was likely to affect cellular Ca2+
homeostasis by targeting plasma membrane voltage-gated Ca2+ channels, as reported by Lucianiet al [32]. NCLX was recently identified as an essential component of the mitochondrial Na+
-Ca2+
exchanger [24], responsible for Ca2+
efflux from mitochondria, thereby providing an opportunity for a specific inhibition of Ca2+efflux from mitochondria through RNA interference. In the present study, silencing of NCLX significantly potentiated depolarisation-induced increases in [Ca2+]
mit(Fig. 6A, B). NCLX silencing also slightly accelerated the onset of the first phase of the [ATP/ ADP]cytresponse to glucose (Fig. 6C, D), but had no significant effect on the amplitude of the [ATP/ADP]cytchanges (Fig. 6C, E).
Chronic glucolipotoxicity inhibits mitochondrial Ca2+ increases and delays [ATP/ADP]cytrecovery
Previous studies [33] have indicated that the structure and localisation of mitochondria are altered in b-cell dysfunction, including glucolipotoxicity, i.e. exposure to high levels of free fatty acids (FFA) and glucose. Importantly, glucose-induced ATP increases in theb-cell are impaired in this model of T2D [34]. We therefore sought to determine whether these changes were also associated with defective mitochondrial Ca2+
increases or altered expression of mitochondrial Ca2+
transporters.
To this end, we cultured primary mouse b-cells under glucolipotoxic conditions (‘‘FFA+
’’ cells) and studied the impact on the dynamics of [Ca2+
]cyt and [Ca2+]mit in response to Vm manipulation. FFA+cells displayed slower dynamics of [Ca2+]
mit increase (Fig. 7A, B). This resulted in a slower onset of the second phase of glucose-induced ATP increase (Fig. 8A, B) in FFA+b
-cells. This effect was not likely to be caused by changes in resting
Ym(213564 mV in controlvs213764 mV in FFA+cells) or the kinetics of the glucose-induced change inYm(Fig. 8C). We also failed to observe any significant change of either MCU or NCLX mRNA levels under these conditions (Fig. 8D). The expression of the transcription factor pancreatic duodenumhomeobox-1 (Pdx1), in contrast, was significantly reduced by the chronic glucolipo-toxicity, in line with earlier observations [35].
Discussion
Multiparametric analysis of glucose signalling in single primaryb-cells
We dissect here the role of mitochondrial Ca2+
transport in the stimulation of single primary pancreaticb-cells with glucose using a combined imaging and electrophysiology approach. This has allowed us to monitor or manipulate up to four key parameters Figure 2. Ca2+entry into the cytosol is essential for the biphasic
increase of cytosolic ATP/ADP.The effect of high glucose on [ATP/ ADP]cytand [Ca2+]cytwas measured in a singleb-cell voltage-clamped at
270 mV (representative of n = 12 cells). Small voltage steps (+5/ 210 mV) were applied every second to measure the slow whole-cell current, Im.Inset: dynamics of [ATP/ADP]cytand Imduring the indicated
simultaneously in the same individual cell. Earlier studies in these cells combined the use of a microelectrode [36] or patch-clamp [37] with [Ca2+
] measurements to report a close association of [Ca2+
]cyt and Vm signals during glucose-induced depolarisation.
Furthermore, the control of Vmusing perforated-patch was shown to be a very efficient means of rapid and precise control of [Ca2+
]cyt[19,38]. The latter strategy provided a powerful tool here to explore the inter-relationships between Ca2+
changes in discrete Figure 3. Mitochondrial [Ca2+] follows the increase in cytosolic [Ca2+] with a delay.A: Colocalisation of 2mt8RP and Mitotracker Orange in a
b-cell, 24 h post infection. B: The effect of 17 mM glucose on Vm, [Ca2+]cyt (Fura-Red) and [Ca2+]mit (2mt8RP) in a single pancreatic b-cell
(representative of n = 10 cells).Inset: Pseudo-colour images of the patched cell cluster presenting pixel-to-pixel ratios at the time points indicated by arrows (i – iii). ROI is indicated with red oval.C: Mean times of maximal increase for [Ca2+
]cytand [Ca2+]mitin pancreaticb-cells, in response to 17 mM
glucose (n = 10 cells). The times are calculated from the moment of the arrival of the first action potential. *Differences are statistically significant (p,0.01).
doi:10.1371/journal.pone.0039722.g003
Ca2+
compartments and with the control of ATP synthesis. Thus, a key technical advantage over earlier studies [14] has been the ability to resolve the exact sequence in which signalling events occurred within the same individual cell. Moreover, possible artefacts resulting from the progressive recruitment of cells within a population were also excluded.
These studies also represent the first use of the novel ATP/ADP probe Perceval [28] in an excitable cell, and provide significant advances over the previous use of less sensitive luciferase-based reporters [7,39]. Although the affinity of Perceval for ATP is relatively high, competition with ADP lowers its sensitivity to a range appropriate for theb-cell cytosol (,1 mM ATP at 3 mM glucose) [7,29]. Importantly, pH changes appeared not to interfere with the probe (Suppl. Fig. S2A).
MCU mediates mitochondrial Ca2+uptake and enhanced ATP synthesis in pancreaticb-cells
We demonstrate here firstly that both cytosolic and mitochon-drial Ca2+
increases are essential for the sustained (second) phase of [ATP/ADP]cyt increase in response to high glucose. Interest-ingly, we show (Fig. 2) that a transient imposed increase in [Ca2+
]cyt is sufficient to lead to a progressive and sustained increase in [ATP/ADP]cyt. This finding is consistent with the possibility that mitochondrial uptake of Ca2+
in response to high glucose (which is slow compared to increases in cytosolic Ca2+
; Fig. 3B, C) may then allow a sustained activation (i.e. ‘‘plasticity’’ or ‘‘memory’’) of oxidative metabolism [39,40].
Recent studies [21,22], have provided convincing evidence for a role of MCU in mitochondrial transport in mammalian fibro-blasts. However, no evidence currently exists demonstrating a role for this protein in this process in a more differentiated cell type. We report here firstly that MCU is critical for mitochondrial Ca2+ accumulation in pancreatic b-cells in response to depolarisation-induced Ca2+
increases. Likewise, we show that the Na+ -Ca2+
exchanger NCLX [24] regulates [Ca2+
]mitincreases and may thus be involved in regulating the responses to glucose, consistent with earlier findings using the pharmacological inhibitor CGP37157 [31]. Specifically, NCLX silencing affected the kinetics of the glucose-induced ATP/ADP changes but had no significant effect on the steady-state ATP/ADP level. Although the mechanisms underlying this unexpected observation are presently unclear, they may involve glucose-dependent changes in cytosolic [Na+
] (unpublished observation of I.S.). Future studies are required to address this question and the role of NCLX in theb-cell.
Overall, our data support a two-phase model (Fig. 9), in which an initial increase in cytosolic [ATP/ADP] (first phase) occurs independently of any increase in cytosolic (or mitochondrial) Ca2+ concentration. In the second phase, the elevation of cytosolic Ca2+ concentration leads to a gradual increase in mitochondrial Ca2+ (Fig. 3B). This, in turn, is likely to activate intramitochondrial dehydrogenases [10] (and perhaps other mitochondrial enzymes) [41], stimulating respiratory chain activity and hence mitochon-drial ATP production. In line with this view, the initial rapid glucose-induced increase in [ATP/ADP]cyt(first phase) was not affected by the MCU silencing whereas the second phase of [ATP/ADP]cytincrease was essentially eliminated.
A recent study [14] also described biphasic increases in cytosolic ATP/ADP in b-cell populations in response to glucose, and indicated that mitochondrial Ca2+accumulation may be essential for increases in cytosolic ATP/ADP in response to the sugar. However, this earlier study relied on the over-expression in the mitochondrial matrix of a high affinity (and high capacity) calcium-binding protein, S100G. Whether the presence of this protein within the mitochondrial matrix may interfere with normal mitochondrial function (for example by leading to a decrease in mitochondrial pH as a result of Ca2+
binding) is unclear. Figure 4. MCU silencing impairs mitochondrial Ca2+ increases.
Pancreaticb-cells were infected with lentiviruses encoding nonsense (‘‘control’’) or anti-MCU (‘‘MCU2’’) shRNA for 72 h. A: [Ca2+]
cyt (Fura-Red) and [Ca2+]mit(2mt8RP) increases were measured in response to 10
depolarising bursts, applied at 4 min21by patch pipette (representative traces for n = 12, control, and n = 10, MCU2cells).B: Mean ratios of maximal
increases in [Ca2+]
mitto the respective increases in [Ca2+]cyt(D[Ca2+]mit/D[Ca2+]cyt) measured in control and MCU2b-cells.
A role for MCU in the regulation ofb-cell excitability and insulin secretion?
Mitochondrial Ca2+
accumulation, catalysed by MCU, is revealed here to be essential for the second phase of glucose-induced ATP synthesis by glucose. What may be the consequences for electrical activity and insulin secretion? Increases in ATP are believed to be involved in both ‘‘KATP-dependent’’ and ‘‘KATP -independent’’ regulation of exocytosis by glucose [16,42]. Impor-tantly, we obtained no evidence for a role for mitochondrial Ca2+ accumulation in the regulation of plasma membrane electrical activity (Fig. 5) suggesting that an involvement of mitochondrial Ca2+in the regulation of insulin secretion, as implied by earlier studies [14], is likely to involve the latter (KATP-independent) action on secretory granule movement or fusion, perhaps powered by ATP increases [43]. Further studies, using larger cell populations, will be necessary to explore the impact of MCU on phasic insulin secretion.
A role for mitochondrial Ca2+ transport inb-cell glucolipotoxicity?
We show here that glucolipotoxic conditions impair Ca2+ transport into mitochondria (Fig. 7) and the second phase of
glucose-induced ATP/ADP increases (Fig. 8). The expression of both MCU and NCLX was unaltered under these conditions (Fig. 8D), in line with previous studies in models of diet-inducedb -cell dysfunction in rodents [44]. It is therefore likely that changes in the intracellular distribution of mitochondria induced by the diabetic milieu [33] are involved in this impairment in mitochon-drial Ca2+
transport. These changes in mitochondrial architecture, and hence localisation at sites of Ca2+
entry into the cytosol [45], may consequently interfere with mitochondrial Ca2+
transport and ATP production.
Conclusions
We show here that mitochondrial Ca2+
uptake in the excitable
b-cell is mediated by MCU and modulated by NCLX. Changes in Ca2+
in the mitochondrial matrix are shown to be critical for increases in cytosolic ATP/ADP ratio, and may thus be required for glucose-stimulated insulin secretion [14]. Manipulation of MCU activity, in particular, may thus provide potential strategies to improve defective insulin secretion in some forms of diabetes. Figure 5. MCU silencing impairs the Ca2+-dependent phase of glucose-induced ATP increase.A
: Glucose-induced changes in Vm, [Ca2+]cyt
and [ATP/ADP]cytwere measured in current clamp, using Fura-Red andPerceval, respectively (representative for n = 8, control, and n = 10, MCU2cells). B: Mean magnitudes of the second phase of [ATP/ADP]cytincrease measured in control and MCU2b-cells. The data were normalised to the width of
the range of [ATP/ADP]cytchange (DFmax), measured as the difference inPercevalfluorescence between the peak point at 17 mM glucose and the
point corresponding to application of 2mM FCCP.C: Changes inDYmmeasured as mitochondrial TMRE fluorescence, in response to the increase of
glucose from 3 to 17 mM, in control and MCU2b-cells. The data are expressed as (F-F
FCCP)/(F0-FFCCP), where F0and FFCCPrepresent TMRE fluorescence
intensity in 3 mM glucose and 2mM FCCP, respectively. *Differences are statistically significant, p,0.01. doi:10.1371/journal.pone.0039722.g005
Ca2+
Figure 6. Effect of the NCLX silencing on [Ca2+]
cytand [Ca2+]mitdynamics.Pancreaticb-cells were infected with lentiviruses delivering
nonsense shRNA (‘‘control’’) or shRNA against NCLX (‘‘NCLX-‘‘) for 36–48 h.A: [Ca2+]
cytand [Ca2+]mitincreases in response to 5 depolarising bursts
applied at 4 min21
were measured using Fura-Red and 2mt8RP, respectively.B: Mean increases in [Ca2+
]mitinduced by a single depolarising burst or
by exposure to 17 mM glucose, related to the respective increases in [Ca2+]
cyt(D[Ca2+]mit/D[Ca2+]cyt).C: Glucose-induced changes in [ATP/ADP]cyt
were measured using Perceval (representative for n = 9 control and n = 9 NCLX2cells).D: Times of half-maximal increase in [ATP/ADP]
cytin response
to 17 mM glucose, in control and NCLX2cells.E: Mean magnitudes of the second phase of [ATP/ADP]
cytincrease measured in control and NCLX2b
-cells. The data were normalised to the width of the range of [ATP/ADP]cytchange (DFmax), measured as the difference inPercevalfluorescence
between the peak point at 17 mM glucose and the point corresponding to application of 2mM FCCP. Differences vs respective NCLX2data are significant with p,0.05 (*) or p,0.01 (**).
Materials and Methods
Islet isolation and culture
Female CD1 mice were sacrificed by cervical dislocation as approved by the United Kingdom Home Office (HO) Animal Scientific Procedures Act, 1986 and designated as ‘‘Schedule 1’’ procedure. Animals were maintained under HO Licence PPL 70/ 7349 (Holder Dr I Leclerc), which received local ethical committee approval, and all participants received approved local training at Imperial College. Pancreatic islets were isolated by collagenase digestion [46], pre-cultured for 5 h in RMPI-1640 medium, containing 11 mM glucose, 10% FCS, 100 U penicillin, 100mg streptomycin, at 37uC, 5%CO2, infected with an appropriate adenovirus encoding cDNA for the required probe, split into single
b-cells and plated on glass coverslips. The cells were then cultured for .24 h in absolute humidity for 2–4 days and assayed as described below. Glass-attached single cells or 2-3-cell clusters displayed an infection efficiency of,90%.b-Cells were identified morphologically and according to their electrophysiological characteristics (membrane capacitance, Vm, KATP current, lack of Na+
current, response to glucose).
Chronic glucolipotoxicity was modelled by culturing the cells in medium containing 0.5 mM Na+
-palmitate and 17 mM glucose for 72 h. Palmitate was prepared as a 150 mM stock in ethanol; the working solution also contained 0.67% fatty-acid free BSA (Sigma). Control medium contained, respectively, 0.67% FFA-free BSA and 0.17% ethanol.
MCU was silenced in primaryb-cells by 24h incubation with shRNA-bearing lentiviral particles (sc-142052-V, Santa-Cruz Biotechnology), at 16106infectious units/ml. Cells infected with
the GFP+control particles (sc-108084) at the same titre displayed a multiplicity of infection of two, 36 hours after infection. Particles delivering non-target shRNA (sc-108080) were used as a negative control.
Molecular biology and generation of adenoviruses cDNA encoding Perceval [28] was excised from pGW1CMV-Perceval plasmid (kindly provided by Prof Gary Yellen, Yale University) by restriction first withEcoRI, then extension using T4 DNA-polymerase and finally by restriction withHindIII to liberate the insert. TheHindIII/blunt insert was cloned into pShuttleCMV previously digested withEcoRV andHindIII.
cDNA encoding 2mt8-ratiometric pericam (2mt8RP) was kindly provided by Prof Tullio Pozzan (University of Padua). ‘‘Mt8’’ refers to the first 36 amino acids of subunit VIII of human cytochromec oxidase (COX) while the targeting efficiency was improved by using two tandem repeats of the addressing sequence [25]. Adenoviral particles were produced as in [47].
Gene expression measurement by qRT-PCR
RNA was purified from islet samples using Trizol. RNA was quantified by Nanodrop spectrophotometer then reverse tran-scribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). mRNA abundance was quantified by qPCR using Sybr Green PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-time PCR machine. Expression of each gene was normalised to cyclophilin A (Ppia), and FFA treatment effect as fold change with 95% confidence intervals was calculated using the
DDCTmethod on 7500 Software (Applied Biosystems, v2.0.5).
Single cell epifluorescence imaging Simultaneous imaging of [Ca2+
] in mitochondria and the cytosol was performed using the mitochondrial pericam 2mt8RP, and Fura-Red (Invitrogen) respectively. 2mt8RP, Fura-Red and Indo-1 were used at single excitation and emission wavelengths. Either dye was dissolved in DMSO (4mM) containing 4% F127-Pluronic. Cells were loaded with Fura-Red by incubation with 4mM of the dye in the extracellular solution for 30 min. Imaging experiments were performed on an Olympus IX-71 microscope with UPlanFL N640 magnification objective. For acquisition, an F-View-II camera and MT-20 excitation system equipped with a Figure 7. Chronic exposure to high-glucose and high-FFA medium impairs Ca2+entry into mitochondria.b
-Cells were pre-cultured in FFA-free medium containing 11 mM glucose (‘‘control’’) or medium containing 17 mM glucose and 0.5 mM palmitate (‘‘FFA+’’) for 48–72 h.A: The
cells were voltage-clamped at270 mV and five depolarising bursts were applied at 4 min21
, as indicated in Vmtrace (above). [Ca2+]cytand [Ca2+]mit
were monitored with Fura-Red and 2mt8RP, respectively. B: Peak [Ca2+]
mitinduced by a single burst related to the respective peak [Ca2+]cyt
(D[Ca2+
]mit/D[Ca2+]cyt), measured in control (blue columns, n = 10) and FFA+(white columns, n = 9) cells. *Differences are significant with p,0.05 (*) or
p,0.01 (**).
doi:10.1371/journal.pone.0039722.g007
Ca2+
Hg/Xe arc lamp were used, under control of CellˆR software (Olympus). Excitation/emission wavelengths were (nm): 410/535 (2mt8RP), 490/630 (Fura-Red), 490/535 (Perceval). Images were acquired at a frequency of 0.2 Hz with typical excitation times of 10 ms. The acquisition of the fluorescence and electrophysiolog-ical data was synchronized using TTL pulses. Imaging data was background-subtracted, analysed and presented as F/F0(Perceval) and F0/F (Fura-Red, 2mt8RP). Whole cells were selected as regions of interest (ROI) to minimize the effect of the cell drift. For cell clusters, only the patched cell was included in the ROI. Every [Ca2+] recording was subjected to the dynamic range control by applying, at the end of the trace, solutions containing 10 mM ionomycin: ‘‘Ca2+-free’’ (0.5 mM EGTA), ‘‘Ca2+-max’’ (5 mM Ca2+). For the [ATP/ADP]
cytrecordings the dynamic range was controlled by high glucose (maximum after.30 min of exposure) and 2mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; minimum).
Measurements of TMRE fluorescence
Cells were loaded with 7 nM TMRE for 60min at 3 mM glucose. Confocal imaging was performed in bath solution (see below) initially containing 3mM glucose, using a Zeiss microscope fitted with a Plan Apochromat x63 n. a. 1.4 oil immersion objective and equipped with Yokogawa CSU22 spinning disk module. The TMRE fluorescence signal was excited at 563 nm using a solid-state laser. Emission at 600 nm was registered using Hamamatsu ImagEM EM-CCD camera. The calculations ofYm were done on the basis of the ratio of mitochondrial and cytosolic fluorescence, as was outlined in [48].
Electrophysiology
Electrophysiological recordings and stimulation were done in whole-cell perforated-patch configuration, using an EPC9 patch-clamp amplifier controlled by Pulse acquisition software (HEKA Elektronik). The pipette tip was dipped into pipette solution, and then back-filled with the same solution containing 0.17 mg/ml amphotericin B. Series resistance and cell capacitance were compensated automatically by the acquisition software. Record-Figure 8. Chronic glucolipotoxicity slows down the second phase of glucose-induced ATP elevation.A: Glucose-induced changes in [ATP/ADP]cytand [Ca2+]cytwere monitored in control (above) and FFA+(below) cells using Perceval and Fura-Red.B: Mean time of saturation of the
second phase of [ATP/ADP]cytincrease in control (blue columns, n = 16) and FFA+(white columns, n = 13) cells.C: Changes inDYmmeasured as
mitochondrial TMRE fluorescence, in response to the increase of glucose from 3 to 17 mM, in control and FFA+b
-cells. The data are expressed as (F-FFCCP)/(F0-FFCCP), where F0and FFCCPrepresent TMRE fluorescence intensity in 3 mM glucose and 2 mM FCCP, respectively.D: Normalised MCU (Ccdc109a), NCLX (Slc24a6) and Pdx1 (Pdx1) mRNA expression levels for control and FFA+
ings, triggered by the TTL pulse, were started in current-clamp mode, and the depolarization of the plasma membrane was monitored simultaneously with [Ca2+
] and [ATP/ADP]cyt, in response to a glucose step from 3 to 17 mM. To monitor the input resistance, the protocol included 10-ms injections of repolarising 10-pA current applied every 20s. The parameters of the current injections were chosen to minimise their effect on the glucose-induced electrical activity. To control Vmand impose electrical stimulations, the mode was periodically switched to voltage-clamp [49]. Vm was held at the value of 270 mV, with 0.5 Hz +5/ 210 mV pulses to monitor the KATP conductance (see Suppl. Fig. S2B). The electrical stimulation was deemed to mimic the naturally occurring bursts of action potentials and comprised of 5-s depolarization trains to230 mV containing 25 ramps of 100 ms to 0 mV and back (Suppl. Fig. S2B). Data were filtered at 1 kHz, and digitised at 2 kHz. Gmwas normalized to cell capacitance to account for cell size.
Experimental solutions
The pipette solution contained (mM): 76 K2SO4, 10 NaCl, 10 KCl, 1 MgCl2, 5 HEPES (pH7.35 with KOH). The extracellular bath solution, referred in text as ‘‘EC’’ contained (mM): 120 NaCl, 4.8 KCl, 24 NaHCO3 (saturated with CO2), 0.5 Na2HPO4, 5 HEPES (pH 7.4 with NaOH), 2.5 CaCl2, 1.2 MgCl2. All experiments were conducted at 32–33uC and the bath solution was perifused continuously.
Data analysis
Imaging data was analysed using CellˆR (Olympus) and ImageJ (Wayne Rasband, NIMH). The simultaneous recordings were combined together and analysed using Igor Pro (Wavemetrics). The results are presented as mean6SEM. Mann-Whitney U-test and Wilcoxon’s paired test were used to assess the statistical significance of the differences between the independent and dependent samples, respectively.
Supporting Information
Figure S1 Expression patterns of Perceval and 2mt8RP.
A: A two-cell pancreaticb-cell cluster was infected with Perceval
(48 h,lex= 490 nm,lem= 535 nm) and incubated with Fura-Red (30 min,lex= 490 nm,lem= 630 nm).B: A three-cell pancreatic
b-cell cluster was infected with 2mt8RP (48 h, lex= 490 nm,
lem= 535 nm) and loaded with Fura-Red (30 min,lex= 490 nm,
lem= 630 nm). (TIF)
Figure S2 Imaging ATP dynamics in single b-cells. Effects of pH, analysis of kinetics. A: Comparison of the
effects of glucose and pH on the Perceval fluorescence. 17 mM glucose was applied to the cell, followed by 140 mM K+
plus 10mM nigericin solutions of the indicated pH.B: Schematic of the
depolarisation protocol (single burst).C: The first phase of
glucose-induced [ATP/ADP]cyt increase and the decrease in Gm were closely associated in time. Gm was calculated from Im traces (Fig. 2B, inset). The pairs of signals (n = 12) were normalised by the range of change during the first phase of ATP elevation. (TIF)
Acknowledgments
We thank Profs. G Yellen (Yale), R.M Denton and P.J. Cullen (Bristol) for useful discussion.
Author Contributions
Conceived and designed the experiments: GAR AIT. Performed the experiments: AIT FS MAR EAB TJP. Analyzed the data: AIT GAR. Contributed reagents/materials/analysis tools: PG RR IS. Wrote the paper: AIT GAR. Proofread the manuscript: TJP.
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![Figure 6. Effect of the NCLX silencing on [Ca 2+ ] cyt and [Ca 2+ ] mit dynamics. Pancreatic b-cells were infected with lentiviruses delivering nonsense shRNA (‘‘control’’) or shRNA against NCLX (‘‘NCLX - ‘‘) for 36–48 h](https://thumb-eu.123doks.com/thumbv2/123dok_br/16437671.196496/8.918.91.674.92.874/figure-silencing-dynamics-pancreatic-infected-lentiviruses-delivering-nonsense.webp)
